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Fetal Bovine Serum (FBS) is the ideal cell growth supplement and generally preferred over other types of cell culture sera. Its high levels of nutrients and optimal combination of growth factors makes Fetal Bovine Serum the most effective cell growth promoter for virtually any cell culture system. In addition, the significantly lower content of antibodies compared to adult and newborn sera minimizes the risk of antibody cross reaction with cells in culture.

2. What is the difference between Fetal Bovine Serum (FBS) and Fetal Calf Serum (FCS)?

Fetal Bovine Serum (FBS) was once known as Fetal Calf Serum (FCS). They are one and the same thing.

3. What is the origin of the serum?

Serum origin should mean the country in which the raw blood was collected. This is not to be confused with the country in which the raw serum was sterile filtered having originated elsewhere. The country of origin should not be a secret when purchasing serum. The documentation from your serum supplier should clearly state the country or countries of origin.

4. Is origin important?

The United States Department of Agriculture (USDA) considers certain countries to be free of bovine spongiform encephalopathy (BSE aka Mad Cow Disease) and foot and mouth disease (FMD). Some of these countries are New Zealand, Australia, and all countries in Central America. However, the USDA permits only serum of New Zealand and Canada origin to be imported into the US without safety testing. Serum of Australian, Mexican, and Central American origin must be quarantined, sampled, and tested by the USDA for the presence of exotic strains of Bluetongue virus and, in the case of Australian serum, Akabane virus as well. Once the quarantined serum has successfully completed safety testing, it can be sold and used in the US without restriction.

There are many areas of the world such as in South America where serum is prohibited from entering the US. Verification of origin is critical for serum users who are producing products requiring regulatory approval through the United States Food and Drug Administration (FDA), the USDA, or other regulatory agencies.

5. Are source and origin the same?

Yes. The source or origin of the serum products refers to the country in which the raw blood was collected. Source or origin does not refer to the country where the raw serum was sterile filtered although collection and filtration may occur in the same country. These terms are frequently misused and confused in the same business.

6. What is the difference between USDA and U.S. Origin FBS?

USDA safety tested FBS is collected in countries such as Mexico and Central America which have been approved by the USDA for bovine serum importation into the United States. Typically, the material is quarantined, sampled, and tested by the USDA prior to release. Once approved material passes test by the USDA, the material is considered legal for sale and use. U.S. Origin FBS comes from USDA licensed abattoirs located entirely within the continental U.S.

7. How is a country’s BSE risk status determined?

The World Organization for Animal Health (OIE) Scientific commission is responsible to undertake, on behalf of the World Assembly, the assessment OIE Member Countries applications for their compliance with OIE standards. The BSE status of a country or zone is determined on the basis of an overall assessment of risk. The two risk categories are Negligible BSE risk and Controlled BSE risk.

8. Do prices vary between fetal bovine serum products from different origins?

Yes, serum is a commodity product and varies among different regions.

Australia and New Zealand origin FBS are generally more expensive than other regions because of the low supply and high collection cost. In addition, two countries are considered to be "quarantined" from the rest of the world (they are islands with no importation of cattle from outside) and are perceived to be more pure because the sera is free from diseases that may be found in other countries.

9. Is traceability of serum to its origin important?

Traceability is the ability of the serum supplier to provide a verifiable document trail from the serum received to the geographic origin of the raw blood used in its preparation. It is critical to the safe use of serum. Issues related to BSE and FMD, for example, can only be controlled by use of serum locations where BSE and FMD do not exist.

10. Is the method of collection important?

Controlling the initial collection of bovine blood is at least as important to serum quality as sterile filtration and aseptic packaging. A closed system collection method (cardiac or venipuncture) and rapid processing are essential to a quality finished serum product. Low levels of endotoxin and hemoglobin are an excellent measure of the care with which collection and processing were done.

11. Is a calf’s age at the time of collection important?

It could be, depending on the application. Blood sourced from an older animal will have different characteristics than blood sourced a younger animal. Also, the type of feed will make a difference (eg. Formula fed veal animals vs. range fed animals). Some differences will be seen when comparing IgG, total protein, and albumin levels (to name a few). The ages of the cattles are listed below:

DescriptionAge

Fetal Bovine Serum Fetus

Newborn Calf Serum Less than 14 days of age

Formula-Fed Calf Serum Less than 22 weeks old

Bovine Calf Serum 14 days to 12 months

Bovine Serum (Adult) 12 months or older

12. Why is lot size important?

Serum is a natural product resulting in lot-to-lot variability. Large lot sizes tend to minimize this variability. Additionally, the cost per liter of lot QC testing prior to purchase is reduced with large lots. Serum suppliers whose lot sizes are large (2,000 to 3,000 liters) will provide the best opportunity of providing consistency order after order.

13. Why is there lot-to-lot variability in serum?

Serum is fundamentally important to the proliferation and maintenance of cells cultured in a classical cell culture medium, as the medium usually will not support cell survival without supplementation with animal serum – typically Fetal Bovine Serum - or human serum, at a concentration of 5-20%. Serum is a complex mixture of biological constituents, a majority of which have not yet been fully defined. The composition of these serum components naturally varies somewhat from lot-to-lot.

14. Why is the filtration method important?

Serum is filtered using a variety of filter types. Common final filters used in serum processing have pore-size ratings ranging from .04 µm to .45 µm and may be used as a single filter or in a series. The industry standard is triple 0.1 µm filters.

15. Why is serum heat inactivated?

Generally, the objective of heat inactivation is to destroy complement activity in the serum without affecting the growth-promoting characteristics of the product. Removal of complement activity from the serum is not required for most cell cultures, but may be necessary for cultures that are sensitive to the complement activity. Since heat inactivation of the serum may, to some extent, decrease the growth performance properties of the serum, this procedure should only be performed if actually required for optimal cell growth. If heat inactivation is required, the process should be carefully controlled to avoid increased denaturation of serum proteins and formation of crystalline precipitates, potentially resulting in excessive loss of growth performance.

Initially, heat inactivation was also used to inactivate microbial contaminants such as mycoplasma. Heat inactivation for this reason is unnecessary, since FBS is triple 0.1 µm filtered and the sera products must test negative for mycoplasma, bacteria and fungi.

Sometimes, heat inactivation is performed to disrupt susceptible viruses. In most protocols for this application, prolonged heat inactivation is required. This is not recommended, since valuable components of the serum are rendered ineffective by this treatment.

16. Should I use heat inactivated serum?

Many laboratories heat inactivate their serum before cell culturing, because it is specified in their protocol. In fact, for many cell culture applications, heat inactivation may be unnecessary. Furthermore, warming the serum-containing medium to 37°C prior to use, as is the practice in many cell culture laboratories, is often sufficient to inactivate heat-labile complement factors.

Heat inactivation, in general, decreases the growth promoting properties of the serum and causes increased formation of crystalline or flocculent precipitates (salts, proteins, lipids, fibrin…) in the serum.

Since every cell type has different growth requirements, the choice of whether to heat inactivate serum or not must be made by the researcher. If there are concerns about whether to use heat inactivated serum with the cell line, perform a side-by-side comparison of cell growth with heat inactivated versus non-heat inactivated serum. Excessive heat inactivation of serum should be avoided whenever possible. If heat inactivation is required, closely monitor the process to avoid overheating the serum or heating over an extended length of time.

When heat inactivation is performed improperly (contents of the bottle are not properly mixed before and during the heat inactivation, serum is heated at temperatures above 56°C or for more than 30 minutes, usage of non-circulating water bath, water level not up to the level of the serum,…), a much greater amount of precipitates will form and the growth promoting ability of the serum will be further compromised.

17. What is gamma irradiated fetal bovine serum?

Gamma irradiation is recognized as a minimally intrusive, effective and reproducible method of inactivating viruses and other adventitious agents in serum. Even though Fetal Bovine Serum is stringently tested, low levels of common bovine viruses below the detection limits of current test methods may exist. Gamma Irradiated Fetal Bovine Serum is exposed to a Cobalt-60 source at a delivered dose of 25-35 kGy under tightly controlled conditions. Exposure of serum to this dosage has been shown to remove up to 6 logs of many extraneous agents while preserving the growth performance characteristics of serum.

18. What lower cost options are available to FBS?

Iron Supplemented Calf Serum product is available. This product is a combination of calf serum formulated with iron to yield a cost effective alternative to FBS and can outperform FBS in a variety of cell culture applications.

19. Is serum light-sensitive?

Serum does contain components that are affected by exposure to light. The effect of light on the serum will depend upon the intensity of light, the wavelength of light and the duration of exposure. Normal handling of serum under room lights should have little effect on the serum. High intensity lights should be avoided and long-term storage under light should be avoided. Certain cell lines may be more sensitive to the effects of light on serum than other cell lines.

20. What is serum turbidity?

Serum turbidity is usually caused by the cryoprecipitation of lipid components during freezing and thawing the serum. The more times the serum is subjected to freeze/thaw cycles, the more turbidity is noticed. Shipping the serum frozen on dry ice at approx (-60) deg C and subsequently storing frozen at (-20) deg C is thermodynamically equivalent to a freeze thaw, even if the product is not allowed to liquefy.

Serum tubidity can be minimized by refraining from repeated freezing and thawing. If serum is not being used all at once, we suggest aliquotting into usable volumes, freezing at (-20) deg C and thawing the aliquots individually at the time of use.

21. How should serum be stored?

Serum is most stable when stored frozen until needed. The recommended storage temperature for serum is between -5°C and -20°C. Repeated freeze-thaw cycles should be avoided as they will cause a decrease in serum performance and an increase in insoluble precipitates. Once a serum is thawed, it is recommended to use the serum the same day or to divide it into single-use aliquots and refreeze it.

22. How to thaw serum to ensure the performance is not compromised?

Frozen serum should be thawed slowly to prevent degradation of the serum.

Remove the serum from frozen storage and thaw at room temperature; or can be refrigerated overnight and completed at room temperature. Periodically agitate the bottle during the thawing process to re-suspend the viscous solutes and to avoid the formation of salt, protein and lipid gradients that can lead to excessive precipitation. Promptly remove the serum from the water bath as soon as the serum is completely thawed. Thoroughly mix the thawed serum before it is added to a culture medium or is heat inactivated as serum components such as proteins and salts tend to concentrate at the bottom of the container during thawing.

Thawing of serum at temperatures above 37°C is not recommended. This process may degrade heat labile nutrients, thus compromising the integrity and performance of the product, and can cause increased precipitate formation.

Once serum is thawed, it should be used promptly or divided into single-use aliquots and refrozen. Alternatively, unused portions of thawed serum may be stored at refrigerated temperatures (2 to 8°C) for a short period of time. However, the length of time that serum can be stored refrigerated with acceptable cell culture results varies with different cell lines.

23. Can serum be used if the product arrives partially thawed?

Serum should be shipped frozen via overnight service and packaged in dry ice. Without a delay in shipping and at the receiving location, it should arrive frozen. If delays occur and the product is partially thawed, the product can still be used. Ensure the bottle is thawed completely, mix gently, aliquot into single-use units (if desired), and refreeze.